Old innovations and shifted paradigms in cellular neuroscience.

Once upon a time the statistics of quantal release were fashionable: "n" available vesicles (fusion sites), each with probability "p" of releasing a quantum. The story was not so simple, a nice paradigm to be abandoned. Biophysicists, experimenting with "black films," explained the astonishing rapidity of spike-induced release: calcium can trigger the fusion of lipidic vesicles with a lipid bilayer, by masking the negative charges of the membranes. The idea passed away, buried by the discovery of NSF, SNAPs, SNARE proteins and synaptotagmin, Munc, RIM, complexin. Electrophysiology used to be a field for few adepts. Then came patch clamp, and multielectrode arrays and everybody became electrophysiologists. Now, optogenetics have blossomed, and the whole field has changed again. Nice surprise for me, when Alvarez de Toledo demonstrated that release of transmitters could occur through the transient opening of a pore between the vesicle and the plasma-membrane, no collapse of the vesicle in the membrane needed: my mentor Bruno Ceccarelli had cherished this idea ("kiss and run") and tried to prove it for 20 years. The most impressive developments have probably regarded IT, computers and all their applications; machine learning, AI, and the truly spectacular innovations in brain imaging, especially functional ones, have transformed cognitive neurosciences into a new extraordinarily prolific field, and certainly let us imagine that we may finally understand what is going on in our brains. Cellular neuroscience, on the other hand, though the large public has been much less aware of the incredible amount of information the scientific community has acquired on the cellular aspects of neuronal function, may indeed help us to eventually understand the mechanistic detail of how the brain work. But this is no more in the past, this is the future.

Selective disruption of synaptic NMDA receptors of the hippocampal trisynaptic circuit in Aß pathology.

Synaptic dysfunction is an early feature in Alzheimer's disease (AD) pathogenesis and a major morphological correlate of memory deficits. Given the main synaptic location of N-methyl-D-aspartate receptors (NMDARs), their dysregulation has been implicated in these pathological effects. Here, to detect possible alterations in the expression and synaptic localisation of the GluN1 subunit in the brain of amyloidogenic APP/PS1 mice, we employed histoblot and SDS-digested freeze-fracture replica labelling (SDS-FRL) techniques. Histoblots showed that GluN1 expression was significantly reduced in the hippocampus in a layer-dependent manner, in the cortex and the caudate putamen of APP/PS1 transgenic mice at 12 months of age but was unaltered at 1 and 6 months. Using quantitative SDS-FRL, we unravelled the molecular organisation of GluN1 in seven excitatory synapse populations at a high spatial resolution in the CA1 and CA3 fields and the DG of the hippocampus in 12-month-old APP/PS1 mice. In the CA1 field, the labelling density for GluN1 in the excitatory synapses established on spines and interneurons, was significantly reduced in APP/PS1 mice compared to age-matched wild-type mice in the stratum lacunosum-moleculare but unaltered in the stratum radiatum. In the CA3 field, synaptic GluN1 was reduced in mossy fibre-CA3 pyramidal cell synapses but unaltered in the A/C-CA3 pyramidal cell synapses. In the DG, the density of GluN1 in granule cell-perforant pathway synapses was reduced in APP/PS1 mice. Altogether, our findings provide evidence of specific alterations of synaptic GluN1 in the trisynaptic circuit of the hippocampus in Aß pathology. This differential vulnerability in the disruption of NMDARs may be involved in the mechanisms causing abnormal network activity of the hippocampal circuit and cognitive impairment characteristic of APP/PS1 mice.

Nanoarchitecture of CaV2.1 channels and GABAB receptors in the mouse hippocampus: Impact of APP/PS1 pathology.

Voltage-gated CaV2.1 (P/Q-type) Ca2+ channels play a crucial role in regulating neurotransmitter release, thus contributing to synaptic plasticity and to processes such as learning and memory. Despite their recognized importance in neural function, there is limited information on their potential involvement in neurodegenerative conditions such as Alzheimer's disease (AD). Here, we aimed to explore the impact of AD pathology on the density and nanoscale compartmentalization of CaV2.1 channels in the hippocampus in association with GABAB receptors. Histoblotting experiments showed that the density of CaV2.1 channel was significantly reduced in the hippocampus of APP/PS1 mice in a laminar-dependent manner. CaV2.1 channel was enriched in the active zone of the axon terminals and was present at a very low density over the surface of dendritic tree of the CA1 pyramidal cells, as shown by quantitative SDS-digested freeze-fracture replica labelling (SDS-FRL). In APP/PS1 mice, the density of CaV2.1 channel in the active zone was significantly reduced in the strata radiatum and lacunosum-moleculare, while it remained unaltered in the stratum oriens. The decline in Cav2.1 channel density was found to be associated with a corresponding impairment in the GABAergic synaptic function, as evidenced by electrophysiological experiments carried out in the hippocampus of APP/PS1 mice. Remarkably, double SDS-FRL showed a co-clustering of CaV2.1 channel and GABAB1 receptor in nanodomains (~40-50 nm) in wild type mice, while in APP/PS1 mice this nanoarchitecture was absent. Together, these findings suggest that the AD pathology-induced reduction in CaV2.1 channel density and CaV2.1-GABAB1 de-clustering may play a role in the synaptic transmission alterations shown in the AD hippocampus. Therefore, uncovering these layer-dependent changes in P/Q calcium currents associated with AD pathology can benefit the development of future strategies for AD management.

Coordinated regulation of phosphatidylinositol 4-phosphate and phosphatidylserine levels by Osh4p and Osh5p is an essential regulatory mechanism in autophagy.

Macroautophagy (hereafter autophagy) is an intracellular degradative pathway in budding yeast cells. Certain lipid types play essential roles in autophagy; yet the precise mechanisms regulating lipid composition during autophagy remain unknown. Here, we explored the role of the Osh family proteins in the modulating lipid composition during autophagy in budding yeast. Our results showed that osh1-osh7∆ deletions lead to autophagic dysfunction, with impaired GFP-Atg8 processing and the absence of autophagosomes and autophagic bodies in the cytosol and vacuole, respectively. Freeze-fracture electron microscopy (EM) revealed elevated phosphatidylinositol 4-phosphate (PtdIns(4)P) levels in cytoplasmic and luminal leaflets of autophagic bodies and vacuolar membranes in all deletion mutants. Phosphatidylserine (PtdSer) levels were significantly decreased in the autophagic bodies and vacuolar membranes in osh4∆ and osh5∆ mutants, whereas no significant changes were observed in other osh deletion mutants. Furthermore, we identified defects in autophagic processes in the osh4∆ and osh5∆ mutants, including rare autophagosome formation in the osh5∆ mutant and accumulation of autophagic bodies in the vacuole in the osh4∆ mutant, even in the absence of the proteinase inhibitor PMSF. These findings suggest that Osh4p and Osh5p play crucial roles in the transport of PtdSer to autophagic bodies and autophagosome membranes, respectively. The precise control of lipid composition in the membranes of autophagosomes and autophagic bodies by Osh4p and Osh5p represents an important regulatory mechanism in autophagy.

GABAB receptors induce phasic release from medial habenula terminals through activity-dependent recruitment of release-ready vesicles.

GABAB receptor (GBR) activation inhibits neurotransmitter release in axon terminals in the brain, except in medial habenula (MHb) terminals, which show robust potentiation. However, mechanisms underlying this enigmatic potentiation remain elusive. Here, we report that GBR activation on MHb terminals induces an activity-dependent transition from a facilitating, tonic to a depressing, phasic neurotransmitter release mode. This transition is accompanied by a 4.1-fold increase in readily releasable vesicle pool (RRP) size and a 3.5-fold increase of docked synaptic vesicles (SVs) at the presynaptic active zone (AZ). Strikingly, the depressing phasic release exhibits looser coupling distance than the tonic release. Furthermore, the tonic and phasic release are selectively affected by deletion of synaptoporin (SPO) and Ca2+-dependent activator protein for secretion 2 (CAPS2), respectively. SPO modulates augmentation, the short-term plasticity associated with tonic release, and CAPS2 retains the increased RRP for initial responses in phasic response trains. The cytosolic protein CAPS2 showed a SV-associated distribution similar to the vesicular transmembrane protein SPO, and they were colocalized in the same terminals. We developed the "Flash and Freeze-fracture" method, and revealed the release of SPO-associated vesicles in both tonic and phasic modes and activity-dependent recruitment of CAPS2 to the AZ during phasic release, which lasted several minutes. Overall, these results indicate that GBR activation translocates CAPS2 to the AZ along with the fusion of CAPS2-associated SVs, contributing to persistency of the RRP increase. Thus, we identified structural and molecular mechanisms underlying tonic and phasic neurotransmitter release and their transition by GBR activation in MHb terminals.

Unique asymmetric distribution of phosphatidylserine and phosphatidylethanolamine in Toxoplasma gondii revealed by nanoscale analysis.

Toxoplasma gondii is a highly prevalent obligate apicomplexan parasite that is important in clinical and veterinary medicine. It is known that glycerophospholipids phosphatidylserine (PtdSer) and phosphatidylethanolamine (PtdEtn), especially their expression levels and flip-flops between cytoplasmic and exoplasmic leaflets, in the membrane of T. gondii play important roles in efficient growth in host mammalian cells, but their distributions have still not been determined because of technical difficulties in studying intracellular lipid distribution at the nanometer level. In this study, we developed an electron microscopy method that enabled us to determine the distributions of PtdSer and PtdEtn in individual leaflets of cellular membranes by using quick-freeze freeze-fracture replica labeling. Our findings show that PtdSer and PtdEtn are asymmetrically distributed, with substantial amounts localized at the luminal leaflet of the inner membrane complex (IMC), which comprises flattened vesicles located just underneath the plasma membrane (see Figs. 2B and 7). We also found that PtdSer was absent in the cytoplasmic leaflet of the inner IMC membrane, but was present in considerable amounts in the cytoplasmic leaflet of the middle IMC membrane, suggesting a barrier-like mechanism preventing the diffusion of PtdSer in the cytoplasmic leaflets of the two membranes. In addition, the expression levels of both PtdSer and PtdEtn in the luminal leaflet of the IMC membrane in the highly virulent RH strain were higher than those in the less virulent PLK strain. We also found that the amount of glycolipid GM3, a lipid raft component, was higher in the RH strain than in the PLK strain. These results suggest a correlation between lipid raft maintenance, virulence, and the expression levels of PtdSer and PtdEtn in T. gondii.

Mapping trasmembrane distribution of sphingomyelin.

Our knowledge on the asymmetric distribution of sphingomyelin (SM) in the plasma membrane is largely based on the biochemical analysis of erythrocytes using sphingomyelinase (SMase). However, recent studies showed that the product of SMase, ceramide, disturbs transmembrane lipid distribution. This led to the development of the complimentary histochemical method, which combines electron microscopy and SM-binding proteins. This review discusses the advantages and caveats of published methods of measuring transbilayer distribution of SM. Recent finding of the proteins involved in the transbilayer movement of SM will also be summarized.

Different modes of synaptic and extrasynaptic NMDA receptor alteration in the hippocampus of P301S tau transgenic mice.

N-methyl-d-aspartate receptors (NMDARs) are pivotal players in the synaptic transmission and synaptic plasticity underlying learning and memory. Accordingly, dysfunction of NMDARs has been implicated in the pathophysiology of Alzheimer disease (AD). Here, we used histoblot and sodium dodecylsulphate-digested freeze-fracture replica labelling (SDS-FRL) techniques to investigate the expression and subcellular localisation of GluN1, the obligatory subunit of NMDARs, in the hippocampus of P301S mice. Histoblots showed that GluN1 expression was significantly reduced in the hippocampus of P301S mice in a laminar-specific manner at 10 months of age but was unaltered at 3 months. Using the SDS-FRL technique, excitatory synapses and extrasynaptic sites on spines of pyramidal cells and interneuron dendrites were analysed throughout all dendritic layers in the CA1 field. Our ultrastructural approach revealed a high density of GluN1 in synaptic sites and a substantially lower density at extrasynaptic sites. Labelling density for GluN1 in excitatory synapses established on spines was significantly reduced in P301S mice, compared with age-matched wild-type mice, in the stratum oriens (so), stratum radiatum (sr) and stratum lacunosum-moleculare (slm). Density for synaptic GluN1 on interneuron dendrites was significantly reduced in P301S mice in the so and sr but unaltered in the slm. Labelling density for GluN1 at extrasynaptic sites showed no significant differences in pyramidal cells, and only increased density in the interneuron dendrites of the sr. This differential alteration of synaptic versus extrasynaptic NMDARs supports the notion that the progressive accumulation of phospho-tau is associated with changes in NMDARs, in the absence of amyloid-ß pathology, and may be involved in the mechanisms causing abnormal network activity of the hippocampal circuit.

Layer-specific distribution and expression pattern of AMPA- and NMDA-type glutamate receptors in the barrel field of the adult rat somatosensory cortex: a quantitative electron microscopic analysis.

AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) and NMDA (N-methyl-d-aspartate) glutamate receptors are driving forces for synaptic transmission and plasticity at neocortical synapses. However, their distribution pattern in the adult rat neocortex is largely unknown and was quantified using freeze fracture replication combined with postimmunogold-labeling. Both receptors were co-localized at layer (L)4 and L5 postsynaptic densities (PSDs). At L4 dendritic shaft and spine PSDs, the number of gold grains detecting AMPA was similar, whereas at L5 shaft PSDs AMPA-receptors outnumbered those on spine PSDs. Their number was significantly higher at L5 vs. L4 PSDs. At L4 and L5 dendritic shaft PSDs, the number of gold grains detecting GluN1 was ~2-fold higher than at spine PSDs. The number of gold grains detecting the GluN1-subunit was higher for both shaft and spine PSDs in L5 vs. L4. Both receptors showed a large variability in L4 and L5. A high correlation between the number of gold grains and PSD size for both receptors and targets was observed. Both receptors were distributed over the entire PSD but showed a layer- and target-specific distribution pattern. The layer- and target-specific distribution of AMPA and GluN1 glutamate receptors partially contribute to the observed functional differences in synaptic transmission and plasticity in the neocortex.

On-grid labeling method for freeze-fracture replicas.

Sodium dodecyl sulfate-treated freeze-fracture replica labeling (SDS-FRL) is an electron microscopic (EM) method that can define the two-dimensional distribution of membrane proteins and lipids in a quantitative manner. Despite its unsurpassed merit, SDS-FRL has been adopted in a limited number of labs, probably because it requires a laborious labeling process as well as equipment and technique for freeze-fracture. Here, we present a method that reduces the manual labor significantly by mounting freeze-fracture replicas on EM grids prior to labeling. This was made possible by the discovery that freeze-fracture replicas invariably adhere to the carbon-coated formvar membrane with their platinum-carbon side, ensuring that the membrane molecules retained in replicas are accessible to labeling solutions. The replicas mounted on EM grids can be stored dry until labeling, checked by light microscopy before labeling and labeled in the same manner as tissue sections. This on-grid method will make SDS-FRL easier to access for many researchers.

ZO-1 Guides Tight Junction Assembly and Epithelial Morphogenesis via Cytoskeletal Tension-Dependent and -Independent Functions.

Formation and maintenance of tissue barriers require the coordination of cell mechanics and cell-cell junction assembly. Here, we combined methods to modulate ECM stiffness and to measure mechanical forces on adhesion complexes to investigate how tight junctions regulate cell mechanics and epithelial morphogenesis. We found that depletion of the tight junction adaptor ZO-1 disrupted junction assembly and morphogenesis in an ECM stiffness-dependent manner and led to a stiffness-dependant reorganisation of active myosin. Both junction formation and morphogenesis were rescued by inhibition of actomyosin contractility. ZO-1 depletion also impacted mechanical tension at cell-matrix and E-cadherin-based cell-cell adhesions. The effect on E-cadherin also depended on ECM stiffness and correlated with effects of ECM stiffness on actin cytoskeleton organisation. However, ZO-1 knockout also revealed tension-independent functions of ZO-1. ZO-1-deficient cells could assemble functional barriers at low tension, but their tight junctions remained corrupted with strongly reduced and discontinuous recruitment of junctional components. Our results thus reveal that reciprocal regulation between ZO-1 and cell mechanics controls tight junction assembly and epithelial morphogenesis, and that, in a second, tension-independent step, ZO-1 is required to assemble morphologically and structurally fully assembled and functionally normal tight junctions.

Alteration in the Synaptic and Extrasynaptic Organization of AMPA Receptors in the Hippocampus of P301S Tau Transgenic Mice.

Tau pathology is a hallmark of Alzheimer's disease (AD) and other tauopathies, but how pathological tau accumulation alters the glutamate receptor dynamics driving synaptic dysfunction is unclear. Here, we determined the impact of tau pathology on AMPAR expression, density, and subcellular distribution in the hippocampus of P301S mice using immunoblot, histoblot, and quantitative SDS-digested freeze-fracture replica labeling (SDS-FRL). Histoblot and immunoblot showed differential regulation of GluA1 and GluA2 in the hippocampus of P301S mice. The GluA2 subunit was downregulated in the hippocampus at 3 months while both GluA1 and GluA2 subunits were downregulated at 10 months. However, the total amount of GluA1-4 was similar in P301S mice and in age-matched wild-type mice. Using quantitative SDS-FRL, we unraveled the molecular organization of GluA1-4 in various synaptic connections at a high spatial resolution on pyramidal cell spines and interneuron dendrites in the CA1 field of the hippocampus in 10-month-old P301S mice. The labeling density for GluA1-4 in the excitatory synapses established on spines was significantly reduced in P301S mice, compared to age-matched wild-type mice, in the strata radiatum and lacunosum-moleculare but unaltered in the stratum oriens. The density of synaptic GluA1-4 established on interneuron dendrites was significantly reduced in P301S mice in the three strata. The labeling density for GluA1-4 at extrasynaptic sites was significantly reduced in several postsynaptic compartments of CA1 pyramidal cells and interneurons in the three dendritic layers in P301S mice. Our data demonstrate that the progressive accumulation of phospho-tau is associated with alteration of AMPARs on the surface of different neuron types, including synaptic and extrasynaptic membranes, leading to a decline in the trafficking and synaptic transmission, thereby likely contributing to the pathological events taking place in AD.

Multiscale modelling of claudin-based assemblies: A magnifying glass for novel structures of biological interfaces.

Claudins (Cldns) define a family of transmembrane proteins that are the major determinants of the tight junction integrity and tissue selectivity. They promote the formation of either barriers or ion-selective channels at the interface between two facing cells, across the paracellular space. Multiple Cldn subunits form complexes that include cis- (intracellular) interactions along the membrane of a single cell and trans- (intercellular) interactions across adjacent cells. The first description of Cldn assemblies was provided by electron microscopy, while electrophysiology, mutagenesis and cell biology experiments addressed the functional role of different Cldn homologs. However, the investigation of the molecular details of Cldn subunits and complexes are hampered by the lack of experimental native structures, currently limited to Cldn15. The recent implementation of computer-based techniques greatly contributed to the elucidation of Cldn properties. Molecular dynamics simulations and docking calculations were extensively used to refine the first Cldn multimeric model postulated from the crystal structure of Cldn15, and contributed to the introduction of a novel, alternative, arrangement. While both these multimeric assemblies were found to account for the physiological properties of some family members, they gave conflicting results for others. In this review, we illustrate the major findings on Cldn-based systems that were achieved by using state-of-the-art computational methodologies. The information provided by these results could be useful to improve the characterization of the Cldn properties and help the design of new efficient strategies to control the paracellular transport of drugs or other molecules.

Different priming states of synaptic vesicles underlie distinct release probabilities at hippocampal excitatory synapses.

A stunning example of synaptic diversity is the postsynaptic target cell-type-dependent difference in synaptic efficacy in cortical networks. Here, we show that CA1 pyramidal cell (PC) to fast spiking interneuron (FSIN) connections have 10-fold larger release probability (Pv) than those on oriens lacunosum-moleculare (O-LM) interneurons. Freeze-fracture immunolabeling revealed that different nano-topologies and coupling distances between Ca2+ channels and release sites (RSs) are not responsible for the distinct Pv. Although [Ca2+] transients are 40% larger in FSINs innervating boutons, when [Ca2+] entry is matched in the two bouton populations, EPSCs in O-LM cells are still 7-fold smaller. However, application of a phorbol ester analog resulted in a ∼2.5-fold larger augmentation at PC - O-LM compared to PC - FSIN synapses, suggesting incomplete docking or priming of vesicles. Similar densities of docked vesicles rule out distinct RS occupancies and demonstrate that incompletely primed, but docked, vesicles limit the output of PC - O-LM synapses.

A Novel Cryogenic Adhesive Retaining Fluidity at Dry-Ice Temperature for Low-Temperature Scanning Electron Microscopy.

Scanning electron microscopy operated at cryogenic temperature (cryo-SEM) is a powerful tool for investigating surface and cross-sectional nanostructures of water-containing samples. Typically, cryo-SEM samples are frozen just before observation in specific metal carriers. However, pre-frozen samples are also of interest, such as frozen food and freeze-stored animal samples. In such cases, sample mounting with a defined orientation is required, but there has been a lack of ideal conductive adhesives that can be used without increasing the sample temperature. Here, we developed a mixture of graphite oxide and 1,3-butanediol as an adhesive, capable of gluing samples at dry-ice temperature and is frozen below that temperature. Dispersion of graphite oxide increased the conductivity and reduced the charge-up contrast. Acquisition of energy-dispersive X-ray spectrum, cross-sectional ion milling, and high-resolution imaging were successfully achieved using the adhesive. We tested and confirmed the usefulness of this new adhesive by applying it to cryo-SEM surface imaging of diatomite, freeze-fractured cross-sectional imaging of chicken liver, and ion milling cross-sectional imaging of a deep-sea snail. The new adhesive is not only useful for food science and field-preserved biological samples but also potentially applicable to wider fields such as archaeological and biological samples preserved under permafrost.

Essential roles of phosphatidylinositol 4-phosphate phosphatases Sac1p and Sjl3p in yeast autophagosome formation.

Autophagy is regulated by phosphoinositides. We have previously shown that phosphatidylinositol 4-phosphate (PtdIns(4)P) is localized in the autophagosomal membrane. Additionally, in yeast cells, phosphatidylinositol 4-kinases Pik1p and Stt4p play important roles in the formation of the autophagosome and its fusion with the vacuole, respectively. In this study, we analyzed the primary role of PtdIns(4)P phosphatases in yeast autophagy. The PtdIns(4)P labeling densities in the membranes of the vacuoles, mitochondria, nucleus, endoplasmic reticulum, and plasma membrane dramatically increased in the phosphatase deletion mutants sac1∆ and sjl3∆, and the temperature-sensitive mutant sac1ts/sjl3∆ at the restrictive temperature. GFP-Atg8 processing assay indicated defective autophagy in the sac1∆ and sac1ts/sjl3∆ mutants. In contrast to the localization of PtdIns(4)P in the luminal leaflet of autophagosomal membranes in the wild-type yeast, PtdIns(4)P was localized in both the luminal and cytoplasmic leaflets of the autophagosomal membranes in the sac1∆ strain. In addition, the number of autophagic bodies in the vacuole significantly decreased in the sac1∆ strain, although autophagosomes were present in the cytoplasm. In the sac1ts/sjl3∆ strain, the number of autophagosomes in the cytoplasm dramatically decreased at the restrictive temperature. Considering that the numbers of autophagosomes and autophagic bodies in the sjl3∆ strain were comparable to those in the wild-type yeast, we found that the autophagosome could not be formed when PtdIns(4)P phosphatase activities of both Sac1p and Sjl3p were diminished. Together, these results indicate that the turnover of PtdIns(4)P by phosphatases is essential for autophagosome biogenesis.

Gold In-and-Out: A Toolkit for Analyzing Subcellular Distribution of Immunogold-Labeled Membrane Proteins in Freeze-Fracture Replica Images.

Integral membrane proteins such as ion channels, transporters, and receptors shape cell activity and mediate cell-to-cell communication in the brain. The distribution, quantity, and clustering arrangement of those proteins contribute to the physiological properties of the cell; therefore, precise quantification of their state can be used to gain insight into cellular function. Using a highly sensitive immunoelectron microscopy technique called sodium dodecyl sulfate-digested freeze-fracture replica immunogold labeling (SDS-FRL), multiple membrane proteins can be tagged with different sizes of immunogold particles at once and visualized two-dimensionally. For quantification, gold particles in the images must be annotated, and then different mathematical and statistical methods must be applied to characterize the distribution states of proteins of interest. To perform such analyses in a user-friendly manner, we developed a program with a simple graphical user interface called Gold In-and-Out (GIO), which integrates several classical and novel analysis methods for immunogold labeled replicas into one self-contained package. GIO takes an input of particle coordinates, then allows users to implement analysis methods such as nearest neighbor distance (NND) and particle clustering. The program not only performs the selected analysis but also automatically compares the results of the real distribution to a random distribution of the same number of particles on the membrane region of interest. In addition to classical approaches for analyzing protein distribution, GIO includes new tools to analyze the positional bias of a target protein relative to a morphological landmark such as dendritic spines, and can also be applied for synaptic protein analysis. Gold Rippler provides a normalized metric of particle density that is resistant to differences in labeling efficiency among samples, while Gold Star is useful for quantifying distances between a protein and landmark. This package aims to help standardize analysis methods for subcellular and synaptic protein localization with a user-friendly interface while increasing the efficiency of these time-consuming analyses.

The Number and Distinct Clustering Patterns of Voltage-Gated Calcium Channels in Nerve Terminals.

Upon the arrival of action potentials at nerve terminals, neurotransmitters are released from synaptic vesicles (SVs) by exocytosis. Ca V 2.1, 2.2, and 2.3 are the major subunits of the voltage-gated calcium channel (VGCC) responsible for increasing intraterminal calcium levels and triggering SV exocytosis in the central nervous system (CNS) synapses. The two-dimensional analysis of Ca V 2 distributions using sodium dodecyl sulfate (SDS)-digested freeze-fracture replica labeling (SDS-FRL) has revealed their numbers, densities, and nanoscale clustering patterns in individual presynaptic active zones. The variation in these properties affects the coupling of VGCCs with calcium sensors on SVs, synaptic efficacy, and temporal precision of transmission. In this study, we summarize how the morphological parameters of Ca V 2 distribution obtained using SDS-FRL differ depending on the different types of synapses and could correspond to functional properties in synaptic transmission.

Electron microscopic visualization of single molecules by tag-mediated metal particle labeling.

Genetically encoded tags have introduced extensive lines of application from purification of tagged proteins to their visualization at the single molecular, cellular, histological and whole-body levels. Combined with other rapidly developing technologies such as clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system, proteomics, super-resolution microscopy and proximity labeling, a large variety of genetically encoded tags have been developed in the last two decades. In this review, I focus on the current status of tag development for electron microscopic (EM) visualization of proteins with metal particle labeling. Compared with conventional immunoelectron microscopy using gold particles, tag-mediated metal particle labeling has several advantages that could potentially improve the sensitivity, spatial and temporal resolution, and applicability to a wide range of proteins of interest (POIs). It may enable researchers to detect single molecules in situ, allowing the quantitative measurement of absolute numbers and exact localization patterns of POI in the ultrastructural context. Thus, genetically encoded tags for EM could revolutionize the field as green fluorescence protein did for light microscopy, although we still have many challenges to overcome before reaching this goal.